FGFR4 antibodies

ABSTRACT

The present invention relates to FGFR4 antibodies including fragments or derivatives thereof and the polynucleotides encoding the antibodies. Expression vectors and host cells comprising the polynucleotides are provided. Further, the invention refers to pharmaceutical compositions comprising the FGFR4 antibodies and methods for the treatment, prevention or diagnosis of disorders associated with FGFR4 expression.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. 371 National Phase Entry Applicationfrom PCT/EP2007/009530, filed Nov. 2, 2007, which claims the benefit ofEuropean Patent Application No. 06022938.2 filed on Nov. 3, 2006, thedisclosure of which is incorporated herein in its entirety by reference.

The present invention relates to FGFR4 antibodies including fragments orderivatives thereof and the polynucleotides encoding the antibodies.Expression vectors and host cells comprising the polynucleotides areprovided. Further, the invention refers to pharmaceutical compositionscomprising the FGFR4 antibodies and methods for the treatment,prevention or diagnosis of disorders associated with FGFR4 expression.

The Fibroblast Growth Factor Receptor 4 (FGFR4) belongs to the family ofFGF receptors which also includes FGFR1, FGFR2 and FGFR3. Like the othermembers of the FGF receptor family, the transmembrane receptor FGFR4consists of an extracellular ligand-binding domain (ECD), atransmembrane domain, an intracellular protein tyrosine kinase domain(TKD) and a C-terminal phosphorylation domain (Klint P et al., 1998).

The FGF receptors are activated by the family of fibroblast growthfactors (FGF), comprising 23 members to date (Eswarakumar et al., 2005;Yamashita, 2005). In contrast to FGFR4, where only two splice variantsare known, other family members such as FGFR1, 2 and 3 can be altered intheir affinity for different FGFs by multiple splice variations (vanHeumen et al., 1999).

FGFR4 is activated by FGF1, FGF2, FGF4, FGF6, FGF8 and FGF9 withdecreasing efficiency (Ornitz et al., 1996); while all of these activatealso other family members, FGF19 is specific for FGFR4 (Xie et al.,1999). Activation of the receptor by FGFs requires binding of the ligandto heparin; interestingly, FGFR4 can also be activated by heparin alone(Gao and Goldfarb, 1995). Many FGFs are broad-spectrum mitogens, whereassome induce cell motility, or alter the state of cellulardifferentiation (for review McKeehan W L, et al., 1998). In vivo, someFGFs have potent angiogenic properties, and others have been implicatedin tissue remodeling, such as that required for wound repair (Werner S,et al., 1994).

Upon binding of the ligand to the extracellular domain of FGFR4,receptor dimerization and subsequent phosphorylation of tyrosine kinaseresidues results in activation of signaling pathways by inducing thebinding of signaling molecules to the receptor (Vainikka et al., 1992);(Vainikka et al., 1994). For example FGFR4 associates with PLC-γ1, andan increase in MAP kinase activation and DNA synthesis upon a FGFstimulation has been observed. Further interaction with other human FGFgrowth factor receptor family members may expand the signaling potentialof FGFR4 and is a means not only for signal diversification but alsosignal amplification (McKeehan W L, & Kan M, 1994). An 85-kDa serinekinase has been found to negatively regulate tyrosine phosphorylation ofFGFR4, but its exact function has not been elucidated (Vainikka et al.,1996). Association of FGFR4 with NCAM has been demonstrated to mediateintegrin-dependent adhesion (Cavallaro et al., 2001), which might play adecisive role in tumor metastasis.

FGFR4 has been reported to inhibit myogenic differentiation (Shaoul etal., 1995), and although muscle development appears to be normal inFGFR4 knockout animals (Weinstein et al., 1998), muscle regenerationafter cardiotoxin-induced damage was shown to be impaired (Zhao et al.,2006). FGFR3/FGFR4 double knockouts are impaired in secondary septationduring alveolus formation, leading to immature lungs (Weinstein et al.,1998). The defect is not observed in FGFR3 knockout mice, which onlyshow skeletal defects. The phenotype of FGFR4 single knockout mice isincreased bile acid synthesis, accompanied by hepatomegaly underhigh-cholesterol diet (Yu et al., 2000).

FGFR4 has been found to be expressed and/or influencing prognosticoutcome in several types of cancer such as melanoma (Streit et al.,2006), breast (Jaakola et al., 1993), prostate (Wang et al., 2004),thyroid (Bernard et al., 2005) and pancreatic cancers (Leung et al.,1994). In addition a polymorphism at position 388 of the polypeptidesequence is associated with a more aggressive disease status in melanoma(Streit et al., 2006), breast (Bange et al, 2002), prostate (Wang etal., 2004), HNSCC (Streit et al., 2004), lung adenocarcinoma (Spinola etal., 2005) and soft tissue sarcoma (Morimoto et al., 2003).

Interestingly, transgenic expression of the FGFR4 specific ligand FGF19under control of a muscle-specific promoter in mice has been found tolead to hepatocellular carcinoma (Nicholes et al., 2002).

Accordingly, agents that interfere with FGFR4 mediated signaling aredesirable. FGFR4 antibodies have been reported, such as in WO 03/063893and WO 99/37299.

An effective strategy to target tumor cells, that is based on thediscovery of the mechanisms of tumor development, is the usage ofmonoclonal antibodies. For example Herceptin™, an antibody directedagainst the receptor tyrosine kinase HER2, improves the median survivalrate of breast cancer patients by approximately 25% compared withchemotherapy alone, and has only very mild side effects. Otherstrategies to use monoclonal antibodies in tumor therapy includeimmunotoxins, like Mylotarg™, a recombinant IgG4 kappa antibodyconjugated to calicheamicin, and antibodies labelled with radioisotopes,as for example Zevalin™.

In order to provide further products for diagnostic and/or therapeuticapplications it is desirable to have FGFR4 antibodies that bindspecifically to the extracellular domain and block FGFR4 mediated signaltransduction.

Thus the technical problem underlying the present invention was toprovide novel FGFR4 antibodies and methods of use of the same which aresuitable for diagnosing, preventing and/or treatment of diseasesassociated with FGFR4 expression.

The solution of the above problems is achieved by providing theembodiments characterized in the claims.

A first aspect of the present invention relates to an antibody includinga fragment or derivative thereof that binds to the extracellular domainof FGFR4, particularly of human FGFR4, and at least partially inhibitsFGFR4 activity.

Preferably, the antibody has at least one antigen binding site, e.g. oneor two antigen binding sites. Further, the antibody preferably comprisesat least one heavy immunoglobulin chain and at least one lightimmunoglobulin chain. An immunoglobulin chain comprises a variabledomain and optionally a constant domain. A variable domain may comprisecomplementary determining regions (CDRs), e.g. a CDR1, CDR2 and/or CDR3region, and framework regions. The term “complementary determiningregion” (CDR) is well-defined in the art (see, for example, Harlow andLane, “Antibodies, a laboratory manual”, CSH Press, Cold Spring Harbour,1988) and refers to the stretches of amino acids within the variableregion of an antibody that primarily makes contact with the antigen.

A second aspect of the present invention relates to an antibodyincluding a fragment or derivative thereof that binds to theextracellular domain of FGFR4 and which comprises at least one heavychain amino acid sequence comprising at least one CDR selected from thegroup consisting of:

-   (a) CDRH1 as shown in SEQ ID NOs: 9 or 15, or a CDRH1 sequence    differing in 1 or amino acids therefrom,-   (b) a CDRH2 as shown in SEQ ID NOs: 10 or 16, or a CDRH2 sequence    differing in 1 or 2 amino acids therefrom, and-   (c) a CDRH3 as shown in SEQ ID NOs: 11 or 17, or a CDRH3 sequence    differing in 1 or 2 amino acids therefrom, and/or    at least one light chain amino acid sequence comprising at least one    CDR selected from the group consisting of:-   (d) a CDRL1 as shown in SEQ ID NOs: 12 or 18, or a CDRL1 sequence    differing in 1 or 2 amino acids therefrom,-   (e) a CDRL2 as shown in SEQ ID NOs: 13 or 19, or a CDRL2 sequence    differing in 1 or 2 amino acids therefrom, and-   (f) a CDRL3 as shown in SEQ ID NOs: 14 or 20, or a CDRL3 sequence    differing in 1 or 2 amino acids therefrom,    or an antibody recognizing the same epitope on the extracellular    domain of FGFR4.

In a preferred embodiment, the antibody comprises at least one heavychain comprising at least one CDR selected from the group consisting of

-   (a) a CDRH1 as shown in SEQ ID NO: 9, or a CDRH1 sequence differing    in 1 or 2 amino acids therefrom,-   (b) a CDRH2 as shown in SEQ ID NO: 10, or a CDRH2 sequence differing    in 1 or 2 amino acids therefrom, and-   (c) a CDRH3 as shown in SEQ ID NO: 11, or a CDRH3 sequence differing    in 1 or 2 amino acids therefrom, and/or    a light chain comprising at least one CDR selected from the group    consisting of-   (d) a CDRL1 as shown in SEQ ID NO: 12, or a CDRL1 sequence differing    in 1 or 2 amino acids therefrom,-   (e) a CDRL2 as shown in SEQ ID NO: 13, or a CDRL2 sequence differing    in 1 or 2 amino acids therefrom, and-   (f) a CDRL3 as shown in SEQ ID NO: 14, or a CDRL3 sequence differing    in 1 or 2 amino acids therefrom, or an antibody recognizing the same    epitope on the extracellular domain of FGFR4.

In a further preferred embodiment, the antibody comprises a heavy chaincomprising at least one CDR selected from the group consisting of

-   (a) a CDRH1 as shown in SEQ ID NO: 15, or a CDRH1 sequence differing    in 1 or 2 amino acids therefrom,-   (b) a CDRH2 as shown in SEQ ID NO: 16, or a CDRH2 sequence differing    in 1 or 2 amino acids therefrom, and-   (c) a CDRH3 as shown in SEQ ID NO: 17, or a CDRH3 sequence differing    in 1 or 2 amino acids therefrom,    and/or a light chain comprising at least one CDR selected from the    group consisting of-   (d) a CDRL1 as shown in SEQ ID NO: 18, or a CDRL1 sequence differing    in 1 or 2 amino acids therefrom,-   (e) a CDRL2 as shown in SEQ ID NO: 19, or a CDRL2 sequence differing    in 1 or 2 amino acids therefrom, and-   (f) a CDRL3 as shown in SEQ ID NO: 20, or a CDRL3 sequence differing    in 1 or 2 amino acids therefrom, or    an antibody recognizing the same epitope on the extracellular domain    of FGFR4.

In another embodiment, the present invention refers to an antibodycomprising a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5 and 7 or at least the variable domainthereof or an amino acid sequence having an identity of at least 90%thereto and/or a light chain amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6 and 8 or at least the variable domainthereof or an amino acid sequence having an identity of at least 90%thereto or to an antibody recognizing the same epitope on theextracellular domain of FGFR4.

In a particular preferred embodiment, the antibody is selected from thegroup consisting of 9A5 and 10F10 or an antibody recognizing the sameepitope on the extracellular domain of FGFR4.

The antibody may be any antibody of natural and/or synthetic origin,e.g. an antibody of mammalian origin. Preferably, the constant domain—ifpresent—is a human constant domain. The variable domain is preferably amammalian variable domain, e.g. a humanized or a human variable domain.More preferably, the antibody is a chimeric, humanized or humanantibody.

The antibody of the invention may be of the IgA-, IgD-, IgE, IgG- orIgM-type, preferably of the IgG- or IgM-type including, but not limitedto, the IgG1-, IgG2-, IgG3-, IgG4-, IgM1- and IgM2-type. In mostpreferred embodiments, the antibody is of the human IgG1-, IgG2- orIgG4-type.

The term antibody includes “fragments” or “derivatives”, which have atleast one antigen binding site of the antibody. Antibody fragmentsinclude Fab fragments, Fab′ fragments F(ab′)₂ fragments as well as Fvfragments. Derivatives of the antibody include single chain antibodies,nanobodies, and diabodies. Derivatives of the antibody shall alsoinclude scaffold proteins having an antibody-like binding activity thatbind to FGFR4.

Within the context of the present invention, the term “scaffoldprotein”, as used herein, means a polypeptide or protein with exposedsurface areas in which amino acid insertions, substitutions or deletionsare highly tolerable. Examples of scaffold proteins that can be used inaccordance with the present invention are protein A from Staphylococcusaureus, the bilin binding protein from Pieris brassicae or otherlipocalins, ankyrin repeat proteins, and human fibronectin (reviewed inBinz and Pluckthun, Curr Opin Biotechnol, 16, 459-69). Engineering of ascaffold protein can be regarded as grafting or integrating an affinityfunction onto or into the structural framework of a stably foldedprotein. Affinity function means a protein binding affinity according tothe present invention. A scaffold can be structurally separable from theamino acid sequences conferring binding specificity. In general,proteins appearing suitable for the development of such artificialaffinity reagents may be obtained by rational, or most commonly,combinatorial protein engineering techniques such as panning againstFGFR4, either purified protein or protein displayed on the cell surface,for binding agents in an artificial scaffold library displayed in vitro,skills which are known in the art (Skerra, J. Mol. Recog., 2000; Binzand Plucktun, 2005). In addition, a scaffold protein having an antibodylike binding activity can be derived from an acceptor polypeptidecontaining the scaffold domain, which can be grafted with bindingdomains of a donor polypeptide to confer the binding specificity of thedonor polypeptide onto the scaffold domain containing the acceptorpolypeptide. The inserted binding domains may include, for example, atleast one CDR of an ant FGFR4 antibody, preferably at least one selectedfrom the group of SEQ ID NOs: 9-20. Insertion can be accomplished byvarious methods known to those skilled in the art including, forexample, polypeptide synthesis, nucleic acid synthesis of an encodingamino acid as well by various forms of recombinant methods well known tothose skilled in the art.

As has been indicated above, the specificity of the antibody, antibodyfragment, a derivative thereof lies in the amino acid sequence of theCDR. The variable domain (the heavy chain VH and light chain VL) of anantibody preferably comprises three complementary determining regionssometimes called hypervariable regions, flanked by four relativelyconserved framework regions or “FRs”. Often, the specificity of anantibody is determined or largely determined by a CDR, such as a CDR ofthe VH chain or a plurality of CDRs. The person skilled in the art willreadily appreciate that the variable domain of the antibody, antibodyfragment or derivative thereof having the above-described CDRs can beused for the construction of antibodies of further improved specificityand biological function. Insofar, the present invention encompassesantibodies, antibody fragments or derivatives thereof comprising atleast one CDR of the above-described variable domains and whichadvantageously have substantially the same, similar or improved bindingproperties as the antibody described in the appended examples. Startingfrom an antibody that comprises at least one CDR as recited in theattached sequence listing and required by the main embodiment of theinvention, the skilled artisan can combine further CDRs from theoriginally identified monoclonal antibodies or different antibodies foran enhanced specificity and/or affinity. CDR grafting is well-known inthe art and can also be used to fine-tune the specific affinity in otherproperties of the antibody, fragment or derivative thereof of theinvention, as long as the original specificity is retained. It isadvantageous that the antibody, fragment or derivative comprises atleast two, more preferred at least three, even more preferred at leastfour such as at least five and particularly preferred all six CDRs ofthe original donor antibody. In further alternatives of the invention,CDRs from different originally identified monoclonal antibodies may becombined in a new antibody entity. In these cases, it is preferred thatthe three CDRs of the heavy chain originate from the same antibodywhereas the three CDRs of the light chain all originate from a different(but all from the same) antibody. The antibodies of the presentinvention or their corresponding immunoglobulin chain(s) can be furthermodified using conventional techniques known in the art, for example, byusing amino acid deletion(s), insertion(s), substitution(s),addition(s), and/or recombination(s) and/or any other modification(s)known in the art either alone or in combination. Methods for introducingsuch modifications in the DNA sequence underlying the amino acidsequence of an immunoglobulin chain are well known to the person skilledin the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual,Cold Spring Harbor Laboratory (1989) N.Y.

The antibodies, antibody fragments or derivative thereof are optionallyde-immunized for therapeutic purposes. The manufacture of de-immunized,e.g. humanized binding proteins may be carried out as described in U.S.Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.

For therapeutic purposes, the antibody may be conjugated with atherapeutic effector group, e.g. a radioactive group or a cytotoxicgroup.

For diagnostic purposes, the antibody may be enzyme labelled. Suitablelabels include radioactive labels, fluorescent labels, or enzyme labels.

The antibody of the invention has advantageous properties with respectto its binding specificity and/or biological activity. Preferably, theFGFR4 antibody exhibits at least one of the following characteristics:

-   -   High specificity for FGFR4, particularly human FGFR4; antibodies        do not significantly recognize other family members, e.g. FGFR1,        FGFR2 and/or FGFR3;    -   Binding to an epitope on the extracellular domain;    -   Blocking or reducing of FGFR4 tyrosine phosphorylation;    -   Blocking or reducing of FGFR4 mediated signal transduction;    -   Decreasing or inhibiting cell growth;    -   Decreasing or inhibiting cell migration.

In a further preferred aspect, the antibody has a constant domain witheffector functions, whereby FGFR4 expressing cells which have bound theantibody, antibody fragment or derivative thereof on the cell surfacemay be attacked by immune system functions. For example, the antibodymay be capable of fixing complement and participating incomplement-dependent cytotoxicity (CDC). Moreover, the antibody may becapable of binding to Fc receptors on effector cells, such as monocytesand natural killer (NK) cells, and participate in antibody-dependentcellular cytotoxicity (ADCC).

As mentioned above and in other words, the antibodies of the inventionshow advantageous properties with respect to their binding specificityand biological activity, in particular with respect to their capacity torecognize epitopes of the FGFR4, to decrease cell growth and cellmigration, the ability to activate a further antineoplastic agent and/orsensitize tumor cells to a therapeutic treatment.

The antibodies of the invention may be obtained by a selection procedurewherein they are tested, e.g. by ELISA, FACS and Western Blot analysisfor their cell binding properties, activities on either signaltransduction pathways or cellular functions and selectivity betweenFGFR4 and the other FGFR family members. In a preferred embodiment theantibody specifically binds to/interacts with at least one epitope ofthe extracellular domain of a mammalian FGFR4, particularly a humanFGFR4, and does not bind to/interact with other FGFR family members. Theterm “extracellular domain” relates to the portion of the FGFR4extending into the extracellular environment. This domain comprisesamino acids 1-360 of the human (?) FGFR4 molecule.

The present invention also encompasses antibodies that compete with theantibodies selected from the group comprising of 9A5 and 10F10 inbinding the same epitope of the extracellular domain of the mammalianFGFR4.

To determine the epitope on FGFR4 recognized by the antibody, chemicallyprepared arrays of protein sequence derived short peptides derived fromthe amino acid sequence of the extracellular FGFR4 domain can be used tolocate and identify antibody epitopes (Reinicke W., Methods Mol. Biol.2004; 248:443-63). A further method to map the epitopes in the FGFR4extracellular domain bound by the antibodies of the invention comprisesSnaps/SELDI (Wang et al., Int J Cancer. 2001 Jun. 15; 92(6):871-6) or aroutine cross-blocking assay such as that described in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed.

Affinity measurements of FGFR4 antibodies of the invention may beperformed by indirect FACS Scatchard analysis. Preferably, this analysiscomprises harvesting an appropriate number of cells of interest, washingwith buffer and seeding on a plate. The cells may be centrifuged toremove supernatant and then resuspended with α-FGFR4 antibody or withantibody dilutions (e.g. 100 μl/well) starting with e.g. 20 μg/mlantibody, diluted e.g. in 1:2 dilution steps. Cell suspensions withantibody are incubated, washed with buffer and incubated with secondaryantibody. The cell suspensions are incubated, washed with buffer andanalyzed (FACS, Beckman Coulter). According to the FACS Scatchardanalysis, the fluorescence mean is calculated for each measurement.Background staining (=without 1^(st) antibody) is subtracted from eachfluorescence mean. Scatchard plot with x-value=fluorescence mean andy-value=fluorescence mean/concentration of antibody generated.

To select for antibodies which reduce ligand induced FGFR4phosphorylation, cells can be preincubated with buffer (control) orantibody, then treated with ligand or control buffer. The cells are thenlysed and the crude lysates can be centrifuged to remove insolublematerial. Supernatants may be incubated with an antibody specific forFGFR4 and protein-A-sepharose to enable efficient precipitation.Following washing, the immunoprecipitates may be separated by SDS-PAGE.Western blots of the gels are then probed with anti-phosphotyrosineantibody. After visualization, the blots may be stripped and re-probedwith an anti-FGFR4 antibody. Reflectance scanning densitometry of thegel can be performed in order to quantify the effect of the antibody inquestion on HRG-induced formation of the complex. Those antibodies whichreduce of FGFR4 phosphorylation relative to control (untreated cells)are selected.

In vitro experiments can be conducted in order to determine the abilityof the antibodies of the invention to inhibit ligand-stimulated cellproliferation. An appropriate number of cells of interests are incubatedwith antibody diluted in appropriate medium. Cells are stimulated byadding ligand directly to antibody solution and are then left to growfor 72 hr. AlamarBlue™ (BIOSOURCE) is added and incubated at 37° C. inthe dark. Absorbance is measured at 590 nm every 30 min.

To select for those antibodies which reduce FGFR4 mediated cellmigration, transmigration experiments can be performed. Serum-starvedcells are incubated with antibody. An appropriate number of cells may beplaced in the top chamber of coated transwells (BD Falcon, 8 μm pores).In the case of stimulation medium alone or containing a chemotacticagent is used in the bottom chamber. Cells are left to migrate and aresubsequently stained. Stained nuclei are counted; percent inhibition isexpressed as inhibition relative to a control antibody.

The effect of the antibody on ligand binding to FGFR4 can be determinedby incubating cells which express this receptor (e.g. MDA-MB 453 breastcancer cells) with radiolabelled ligand (e.g. FGF1 or FGF19), in theabsence (control) or presence of the FGFR4 antibody. Those antibodieswhich reduce the binding affinity of ligand for the FGFR4 receptor orwhich block binding of ligand to FGFR4 can be identified.

The anti-tumor efficacy of therapeutic antibodies may be evaluated inhuman xenograft tumor studies. In these studies, human tumors grow asxenografts in immunocompromised mice and therapeutic efficacy ismeasured by the degree of tumor growth inhibition. In order todetermine, if the FGFR4 antibodies of the invention interfere with tumorgrowth of human cancer cells in nude mice, cells are implanted innude/nude mice. Tumors are subcutaneous, grown on the back of theanimal. Treatment may be started immediately or when tumors reach a meanvolume of 20-50 mm³. Prior to first treatment, mice are randomized andstatistical tests performed to assure uniformity in starting tumorvolumes (mean, median and standard deviation) across treatment groups.Treatment is started with a loading dose of 50 mg/kg followed by 25mg/kg injections once a week by intraperitoneal injection. A control armreceives a known antineoplastic agent, e.g. doxorubicin (pharmaceuticalgrade).

In a preferred embodiment of the invention, the antibody is a monoclonalantibody. Monoclonal antibodies can be prepared, for example, by thewell-established techniques as originally described in Köhler andMilstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981),3, which comprise the fusion of rat myeloma cells to spleen cellsderived from immunized mammals with modifications developed by the art.

In an additionally preferred embodiment of the invention, the antibodymay be a Fab-fragment, a F(ab₂)′-fragment, a single-chain antibody, achimeric antibody, a CDR-grafted antibody, a bivalentantibody-construct, a humanized antibody, a human, a synthetic antibody,or a chemically modified derivative thereof, a multispecific antibody, adiabody, a nanobody, a Fv-fragment, or another type of a recombinantantibody

Fragments or derivatives of the above antibodies directed to theaforementioned epitopes can be obtained by using methods which aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodiesare obtained by the phage display technique, surface plasmon resonanceas employed in the BIAcore system can be used to increase the efficiencyof phage antibodies which bind to an epitope of FGFR4 (Schier, HumanAntibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods183 (1995), 7-13).

The antibodies herein specifically include “chimeric” antibodies(immunoglobulins) in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The production ofchimeric antibodies is described, for example, in WO 89/09622.

Humanized forms of the antibodies may be generated according to themethods known in the art such as chimerization or CDR grafting. Methodsfor the production of humanized antibodies are well known in the art andare described in, e.g., EP-A1 0 239 400 and W090/07861. Generally, ahumanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

A de-immunized antibody is a protein devoid of or reduced for epitopesthat can be recognized by T helper lymphozytes. An example how toidentify said epitopes is shown in Tangri et al., (J Immunol. 2005 Mar.15; 174(6): 3187-96.).

Further antibodies to be utilized in accordance with the presentinvention are so-called xenogenic antibodies. The general principle forthe production of xenogenic antibodies such as human antibodies in miceis described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO96/33735.

As discussed above, the antibody of the invention may exist in a varietyof forms besides complete antibodies; including, for example, Fv, Faband F(ab)₂ as well as in single chains; see e.g. W088/09344.

If desired, the antibodies of the invention may be mutated in thevariable domains of the heavy and/or light chains to alter a bindingproperty of the antibody. For example, a mutation may be made in one ormore of the CDR regions to increase or decrease the Kd of the antibodyfor FGFR4, or to alter the binding specificity of the antibody.Techniques in site directed mutagenesis are well-known in the art. See,e.g., Sambrook et al. and Ausubel et al., supra. Furthermore, mutationsmay be made at an amino acid residue that is known to be changedcompared to germline in a variable region of an FGFR4 antibody. Inanother aspect, mutations may be introduced into one or more of theframework regions. A mutation may be made in a framework region orconstant domain to increase the half-life of the FGFR4 antibody. See,e.g., WO 00/09560. A mutation in a framework region or constant domainmay also be made to alter the immunogenicity of the antibody, to providea site for covalent or non-covalent binding to another molecule, or toalter such properties as complement fixation. Mutations may be made ineach of the framework regions, the constant domain and the variableregions in a single mutated antibody. Alternatively, mutations may bemade in only one of the framework regions, the variable regions or theconstant domain in a single mutated antibody.

The invention further relates to a nucleic acid molecule encoding theantibody, antibody fragment or derivative thereof of the invention. Thenucleic acid molecule of the invention encoding the above-describedantibody, antibody fragment or derivative thereof may be, e.g. DNA,cDNA, RNA or synthetically produced DNA or RNA or recombinantly producedchimeric nucleic acid molecule comprising any of those nucleic acidmolecules either alone or in combination. The nucleic acid molecule mayalso be genomic DNA corresponding to the entire gene or a substantialportion thereof or to fragments and derivatives thereof. The nucleotidesequence may correspond to the naturally occurring nucleotide sequenceor may contain single or multiple nucleotide substitutions, deletions oradditions. In a particular preferred embodiment of the presentinvention, the nucleic acid molecule is a cDNA molecule.

Preferably, the invention relates to an isolated nucleic acid moleculeselected from the group consisting of:

-   (a) a nucleic acid sequence encoding a polypeptide of SEQ ID NOs:    5-20,-   (b) a nucleic acid sequence as shown in SEQ ID NOs: 1-4,-   (c) a nucleic acid complementary to any of the sequences in (a) or    (b); and-   (d) a nucleic acid sequence capable of hybridizing to (a), (b)    or (c) under stringent conditions.

The term “hybridizing under stringent conditions” means that two nucleicacid fragments hybridize with one another under standardizedhybridization conditions as described for example in Sambrook et al.,“Expression of cloned genes in E. coli” in Molecular Cloning: Alaboratory manual (1989), Cold Spring Harbor Laboratory Press, New York,USA. Such conditions are for example hybridization in 6.0×SSC at about45° C. followed by a washing step with 2.0×SSC at 50° C., preferably2.0×SSC at 65° C., or 0.2×SSC at 50° C., preferably 0.2×SSC at 65° C.

The invention also relates to a vector comprising a nucleic acidmolecule of the invention. Said vector may be, for example, a phage,plasmid, viral or retroviral vector. Retroviral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host/cells.

The nucleic acid molecules of the invention may be joined to a vectorcontaining selectable markers for propagation in a host. Generally, aplasmid vector is introduced in a precipitate such as a calciumphosphate precipitate or rubidium chloride precipitate, or in a complexwith a charged lipid or in carbon-based clusters, such as fullerens.Should the vector be a virus, it may be packaged in vitro using anappropriate packaging cell line prior to application to host cells.

Preferably, the vector of the invention is an expression vector whereinthe nucleic acid molecule is operatively linked to one or more controlsequences allowing the transcription and optionally expression inprokaryotic and/or eukaryotic host cells. Expression of said nucleicacid molecule comprises transcription of the nucleic acid molecule,preferably into a translatable mRNA. Regulatory elements ensuringexpression in eukaryotic cells, preferably mammalian cells, are wellknown to those skilled in the art. They usually comprise regulatorysequences ensuring initiation of transcription and optionally poly-Asignals ensuring termination of transcription and stabilization of thetranscript. Additional regulatory elements may include transcriptionalas well as translational enhancers. Possible regulatory elementspermitting expression in prokaryotic host cells comprise, e.g., the lac,trp or tac promoter in E. coli, and examples for regulatory elementspermitting expression in eukaryotic host cells are the AOXI or GAL1promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus),CMV-enhancer, SV40-enhancer or a globin intron in mammalian and otheranimal cells. Beside elements which are responsible for the initiationof transcription such regulatory elements may also comprisetranscription termination signals, such as the SV40-poly-A site or thetk-poly-A site, downstream of the polynucleotide. In this context,suitable expression vectors are known in the art such as Okayama-BergcDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3(Invitrogen) or pSPORTI (GIBCO BRL). Preferably, said vector is anexpression vector and/or a gene transfer or targeting vector. Expressionvectors derived from viruses such as retroviruses, vaccinia virus,adeno-associated virus, herpes viruses, or bovine papilloma virus, maybe used for delivery of the polynucleotides or vector of the inventioninto targeted cell population. Methods which are well known to thoseskilled in the art can be used to construct recombinant viral vectors;see, for example, the techniques described in Sambrook, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory (2001, ThirdEdition) N.Y. and Ausubel, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y. (1994).Alternatively, the nucleic acid molecules of the invention can bereconstituted into liposomes for delivery to target cells.

The invention further relates to a host comprising the vector of theinvention. Said host may be a prokaryotic or eukaryotic cell or anon-human transgenic animal. The polynucleotide or vector of theinvention which is present in the host may either be integrated into thegenome of the host or it may be maintained extrachromosomally. In thisrespect, it is also to be understood that the nucleic acid molecule ofthe invention can be used for “gene targeting” and/or “genereplacement”, for restoring a mutant gene or for creating a mutant genevia homologous recombination; see for example Mouellic, Proc. Natl.Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, APractical Approach, Oxford University Press.

The host can be any prokaryotic or eukaryotic cell, such as a bacterial,insect, fungal, plant, animal, mammalian or, preferably, human cell.Preferred fungal cells are, for example, those of the genusSaccharomyces, in particular those of the species S. cerevisiae. Theterm “prokaryotic” is meant to include all bacteria which can betransformed or transfected with a polynucleotide for the expression of avariant polypeptide of the invention. Prokaryotic hosts may include gramnegative as well as gram positive bacteria such as, for example, E.coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Apolynucleotide coding for a mutant form of variant polypeptides of theinvention can be used to transform or transfect the host using any ofthe techniques commonly known to those of ordinary skill in the art.Methods for preparing fused, operably linked genes and expressing themin bacteria or animal cells are well-known in the art (Sambrook,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory(2001, Third Edition). The genetic constructs and methods describedtherein can be utilized for expression of variant antibodies, antibodyfragments or derivatives thereof of the invention in, e.g., prokaryotichosts. In general, expression vectors containing promoter sequenceswhich facilitate the efficient transcription of the inserted nucleicacid molecule are used in connection with the host. The expressionvector typically contains an origin of replication, a promoter, and aterminator, as well as specific genes which are capable of providingphenotypic selection of the transformed cells. The transformedprokaryotic hosts can be grown in fermentors and cultured according totechniques known in the art to achieve optimal cell growth. Theantibodies, antibody fragments or derivatives thereof of the inventioncan then be isolated from the grown medium, cellular lysates, orcellular membrane fractions. The isolation and purification of themicrobially or otherwise expressed antibodies, antibody fragments orderivatives thereof of the invention may be by any conventional meanssuch as, for example, preparative chromatographic separations andimmunological separations such as those involving the use of monoclonalor polyclonal antibodies.

In a preferred embodiment of the invention, the host is a bacterium,fungal, plant, amphibian or animal cell. Preferred animal cells includebut are not limited to Chinese hamster ovary (CHO) cells, baby hamsterkidney (BHK) cells, monkey kidney cells (COS), 3T3 cells, NSO cells anda number of other cell lines, including human cells. In anotherpreferred embodiment, said animal cell is an insect cell. Preferredinsect cells include but are not limited to cells of the SF9 cell lines

In a more preferred embodiment of the invention, said host is a humancell or human cell line. Said human cells include, but are not limitedto Human embryonic kidney cells (HEK293, 293T, 293 freestyle).Furthermore, said human cell lines include, but are not limited to HeLacells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells.

The invention also provides transgenic non-human animals comprising oneor more nucleic acid molecules of the invention that may be used toproduce antibodies of the invention. Antibodies can be produced in andrecovered from tissue or body fluids, such as milk, blood or urine, ofgoats, cows, horses, pigs, rats, mice, rabbits, hamsters or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690; 5,756,687; 5,750,172; and5,741,957. As described above, non-human transgenic animals thatcomprise human immunoglobulin loci can be produced by immunizing withFGFR4 or a portion thereof.

The invention additionally relates to a method for the preparation of anantibody, comprising culturing the host of the invention underconditions that allow synthesis of said antibody and recovering saidantibody from said culture.

The transformed hosts can be grown in fermentors and cultured accordingto techniques known in the art to achieve optimal cell growth. Onceexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like; see, Scopes, “Protein Purification”,Springer-Verlag, N.Y. (1982). The antibody or its correspondingimmunoglobulin chain(s) of the invention can then be isolated from thegrowth medium, cellular lysates, or cellular membrane fractions. Theisolation and purification of the, e.g., microbially expressedantibodies or immunoglobulin chains of the invention may be by anyconventional means such as, for example, preparative chromatographicseparations and immunological separations such as those involving theuse of monoclonal or polyclonal antibodies directed, e.g., against theconstant region of the antibody of the invention.

It will be apparent to those skilled in the art that the antibodies ofthe invention can be further coupled to other moieties for, e.g., drugtargeting and imaging applications. Such coupling may be conductedchemically after expression of the antibody or antigen to site ofattachment or the coupling product may be engineered into the antibodyor antigen of the invention at the DNA level. The DNAs are thenexpressed in a suitable host system, and the expressed proteins arecollected and renatured, if necessary.

In a preferred embodiment of the present invention, the antibody iscoupled to an effector, such as a radioisotope or a toxicchemotherapeutic agent. Preferably, these antibody conjugates are usefulin targeting cells, e.g. cancer cells, expressing FGFR4, forelimination. The linking of antibodies/antibody fragments of theinvention to radioisotopes e.g. provides advantages to tumor treatments.Unlike chemotherapy and other forms of cancer treatment,radioimmunotherapy or the administration of a radioisotope-antibodycombination directly targets the cancer cells with minimal damage tosurrounding normal, healthy tissue. Preferred radioisotopes include e.g.³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

Furthermore, the antibodies of the invention can be used to treat cancerwhen being conjugated with toxic chemotherapeutic drugs such asgeldanamycin (Mandler et al., J. Natl. Cancer Inst., 92(19), 1549-51(2000)) and maytansin, for example, the maytansinoid drug, DM1 (Liu etal., Proc. Natl. Acad. Sci. U.S.A. 93:8618-8623 (1996) and auristatin-Eor monomethylauristatin-E (Doronina et al., Nat. Biotechnol. 21:778-784(2003) or calicheamicin. Different linkers that release the drugs underacidic or reducing conditions or upon exposure to specific proteases areemployed with this technology. The antibodies of the invention may beconjugated as described in the art.

The invention further relates to a pharmaceutical composition comprisingthe antibody, the nucleic acid molecule, the vector, the host of theinvention or an antibody obtained by the method of the invention.

The term “composition” as employed herein comprises at least onecompound of the invention. Preferably, such a composition is apharmaceutical or a diagnostic composition.

It is preferred that said pharmaceutical composition comprises apharmaceutically acceptable carrier and/or diluent. The herein disclosedpharmaceutical composition may be partially useful for the treatment ofdisorders associated with, accompanied by or caused by EGFR4 expression,overexpression or hyperactivity, e.g. hyperproliferative diseases,inflammatory diseases or metabolic diseases. Said disorders comprise,but are not limited to psoriasis, obesity, cancer, e.g. breast, lung,colon, kidney, lymphoma, skin, ovary, prostate, pancreas, esophagus,barret, stomach, bladder, cervix, liver, thyroid cancer, melanoma, orother hyperplastic or neoplastic diseases or other FGFR4 expressing oroverexpressing diseases.

Examples of suitable pharmaceutical carriers, excipients and/or diluentsare well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions etc. Compositions comprising suchcarriers can be formulated by well known conventional methods. Thesepharmaceutical compositions can be administered to the subject at asuitable dose. Administration of the suitable compositions may beeffected by different ways, e.g., by intravenous, intraperitoneal,subcutaneous, intramuscular, topical, intradermal, intranasal orintrabronchial administration. The compositions of the invention mayalso be administered directly to the target site, e.g., by biolisticdelivery to an external or internal target site, like the brain. Thedosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. Proteinaceous pharmaceutically active mattermay be present in amounts between 1 μg and 100 mg/kg body weight perdose; however, doses below or above this exemplary range are envisioned,especially considering the aforementioned factors. If the regimen is acontinuous infusion, it should also be in the range of 1 pg to 100 mgper kilogram of body weight per minute.

Progress can be monitored by periodic assessment. The compositions ofthe invention may be administered locally or systemically. Preparationsfor parenteral administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's, or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.Furthermore, the pharmaceutical composition of the invention maycomprise further agents depending on the intended use of thepharmaceutical composition. It is particularly preferred that thepharmaceutical composition comprises further active agents like, e.g. anadditional antineoplastic agent, small molecule inhibitor, anti-tumoragent or chemotherapeutic agent.

The invention also relates to a pharmaceutical composition comprisingthe antibody of the invention in combination with at least one furtheranti-neoplastic agent. Said combination is effective, for example, ininhibiting abnormal cell growth.

Many antineoplastic agents are presently known in the art. In oneembodiment, the antineoplastic agent is selected from the group oftherapeutic proteins including but not limited to antibodies orimmunomodulatory proteins. In another embodiment the antineoplasticagent is selected from the group of small molecule inhibitors orchemotherapeutic agents consisting of mitotic inhibitors, kinaseinhibitors, alkylating agents, anti-metabolites, intercalatingantibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes,topoisomerase inhibitors, histone deacetylase inhibitors, anti-survivalagents, biological response modifiers, anti-hormones, e.g.anti-androgens, and antiangiogenesis agents.

In yet another embodiment the pharmaceutical composition comprises theantibody and a an inhibitor of a member of the EGFR family, e.g. anEGFR, HER2, HER3 or HER4 inhibitor, particularly a HER2 inhibitor, e.g.an antagonistic antibody or a small molecule inhibitor.

The pharmaceutical composition of the invention can be used in humanmedicine and can be used also for veterinary purposes.

Additionally, the invention relates to the use of the antibody of theinvention, the nucleic acid molecule, the vector, the host of theinvention or an antibody obtained by the method of the invention for thepreparation of a pharmaceutical composition for diagnosis, prevention ortreatment of hyperproliferative diseases, inflammatory diseases ormetabolic diseases, particularly of disorders associated with,accompanied by or caused by FGFR4 expression, overexpression orhyperactivity.

A hyperproliferative disease as mentioned above includes any neoplasia,i.e. any abnormal and/or uncontrolled new growth of tissue. The term“uncontrolled new growth of tissue” as used herein may depend upon adysfunction and/or loss of growth regulation. A hyperproliferativedisease includes tumor diseases and/or cancer, such as metastatic orinvasive cancers.

In a preferred embodiment of the use of the invention, saidhyperproliferative disease is in particular breast, lung, colon, kidney,lymphoma, skin, ovary, prostate, pancreas, esophagus, barret, stomach,bladder, cervix, liver, thyroid cancer, melanoma, hyperplastic orneoplastic diseases or other FGFR4 expressing or overexpressinghyperproliferative diseases.

In yet another embodiment the present invention relates to a diagnosticcomposition comprising the antibody of the invention, the nucleic acidmolecule, the vector, the host of the invention or an antibody obtainedby the method of the invention and optionally a pharmaceuticallyacceptable carrier.

The diagnostic composition of the invention is useful in the detectionof an undesired expression, overexpression or hyperactivity of themammalian FGFR4 in different cells, tissues or another suitable sample,comprising contacting a sample with an antibody of the invention, anddetecting the presence of FGFR4 in the sample. Accordingly, thediagnostic composition of the invention may be used for assessing theonset or the disease status of a hyperproliferative disease.

Furthermore, malignant cells, such as cancer cells expressing FGFR4, canbe targeted with the antibody of the invention. The cells which havebound the antibody of the invention might thus be attacked by immunesystem functions such as the complement system or by cell-mediatedcytotoxicity, therefore reducing in number of or eradicating cancercells. These considerations equally apply to the treatment of metastasesand re-current tumors.

In another aspect of the present invention, the antibody of theinvention is coupled to a labelling group. Such antibodies areparticularly suitable for diagnostic applications. As used herein, theterm “labelling group” refers to a detectable marker, e.g. aradiolabelled amino acid or biotinyl moieties that can be detected bymarked avidin. Various methods for labelling polypeptides andglycoproteins, such as antibodies, are known in the art and may be usedin performing the present invention. Examples of suitable labellinggroups include, but are not limited to, the following: radioisotopes orradionuclides (e.g. 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I),fluorescent groups (e.g. FITC, rhodamine, lanthanide phosphors),enzymatic groups (e.g. horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase), chemiluminescent groups, biotinylgroups, or predetermined polypeptide epitopes recognized by a secondaryreporter (e.g. leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags).

In certain aspects, it may be desirable, that the labelling groups areattached by spacer arms of various lengths to reduce potential sterichindrance.

The above embodiment of the invention is particularly important. Sincethe antibodies of the invention show a broad scope of applicability withrespect to different mammalian species that can be treated, thediagnostic composition of the invention is also useful and applicable indifferent mammalian species.

In another embodiment the present invention relates to a method ofassessing for the presence of FGFR4 expressing cells comprisingcontacting the antibody of the invention with cells or a tissuesuspected of carrying FGFR4 on their/its surface. Suitable methods fordetection of FGFR4 expression in a sample may be an Enzyme-LinkedImmunosorbent Assay (ELISA) or Immunohistochemistry (IHC).

An ELISA assay may be carried out in a microtiter plate format, whereine.g. wells of a microtiter plate, are adsorbed with a FGFR4 antibody.The wells are rinsed and treated with a blocking agent such as milkprotein or albumin to prevent nonspecific adsorption of the analyte.Subsequently the wells are treated with a test sample. After rinsingaway the test sample or standard, the wells are treated with a secondFGFR4 antibody that is labelled, e.g. by conjugation with biotin. Afterwashing away excess secondary antibody, the label is detected, e.g. withavidin-conjugated horseradish peroxidase (HRP) and a suitablechromogenic substrate. The concentration of the FGFR4 antigen in thetest samples is determined by comparison with a standard curve developedfrom standard samples.

For IHC, paraffin-embedded tissues may be used, wherein the tissues are,e.g. first deparaffinized in xylene and then dehydrated, e.g. withethanol and rinsed in distilled water. Antigenic epitopes masked byformalin-fixation and paraffin-embedding may be exposed by epitopeunmasking, enzymatic digestion or saponin. For epitope unmaskingparaffin sections may be heated in a steamer, water bath or microwaveoven for 20-40 min in a epitope retrieval solution as for example 2N HClsolution (pH 1.0). In the case of an enzyme digestion, tissue sectionsmay be incubated at 37° C. for 10-30 minutes in different enzymesolutions such as proteinase K, trypsin, pronase, pepsin etc.

After rinsing away the epitope retrieval solution or excess enzyme,tissue sections are treated with a blocking buffer to prevent unspecificinteractions. The primary FGFR4 antibody is added at appropriateconcentrations. Excess primary antibody is rinsed away and sections areincubated in peroxidase blocking solution for 10 min at roomtemperature. After another washing step, tissue sections are incubatedwith a secondary labelled antibody, e.g. labelled with a group thatmight serve as an anchor for an enzyme. Examples therefore are biotinlabelled secondary antibodies that are recognized by streptavidincoupled horseradish peroxidase. Detection of the antibody/enzyme complexis achieved by incubating with a suitable chromogenic substrate.

In an additional embodiment the present invention relates to a method ofblocking FGFR4 function comprising contacting the antibody of theinvention with cells or a tissue suspected of carrying FGFR4 ontheir/its surface under conditions, wherein the antibody is capable ofblocking FGFR4 function. The contacting may be in vitro or in vivo.

The invention also relates to a method of treating a hyperproliferativedisease, a metabolic disease or an inflammatory disease comprisingadministering to a patient in need thereof a suitable dose of theantibody or antibody fragment or derivative thereof of the presentinvention. The hyperproliferative disease is preferably selected fromdisorders associated with, accompanied by or caused by EGFR4 expression,overexpression or hyperactivity, such as cancer, e.g. breast, lung,colon, kidney, lymphoma, skin, ovary, prostate, pancreas, esophagus,barret, stomach, bladder, cervix, liver, thyroid cancer and hyperplasticand neoplastic diseases or other FGFR4 expressing or overexpressinghyperproliferative diseases.

The invention further relates to a method of treating a disease whereinthe antibody of the invention is administered to a mammal and whereinsaid disease is correlated directly or indirectly with the abnormallevel of expression or activity of FGFR4.

Finally, the invention relates to a kit comprising the antibody,antibody fragment or derivative thereof of the invention, the nucleicacid molecule encoding said components and/or the vector of theinvention.

All embodiments covering the compounds disclosed herein can be used assingle compounds or in combination for the preparation of a medicament.

FIGURE LEGENDS

FIG. 1. Anti-FGFR4 antibodies 10F10 and 9A5 do not bind to the othermembers of the family, FGFR1-3. Extracellular domains of FGFR1-4 wereexpressed as myc-tagged recombinant proteins. Antibody binding torecombinant proteins or BSA was detected by ELISA, using an anti-mycantibody to control for equivalent coating.

FIG. 2. Determination of 10F10 antibody affinities in FACS Scatchardexperiments on L6-FGFR4 cells.

FIG. 3. Anti-FGFR4 antibodies 10F10 and 9A5 inhibit FGF1-induced FGFR4tyrosine phosphorylation. L6-FGFR4 cells were starved, incubated withantibodies for 1 hour and stimulated with 10 ng FGF1 for 10 minutes.FGFR4 was precipitated from cell lysates with antibody C-19 and theWestern blot probed with anti-phospho-tyrosine antibody 4G10 andre-probed with C-19.

FIG. 4. Anti-FGFR4 antibodies 10F10 and 9A5 inhibit Erk phosphorylationstimulated by FGF1 or FGF19 in FGFR4-transfected L6 myoblasts. L6 cellsstably transfected with FGFR4 were starved, incubated with FGFR4antibodies 10F10 or 9A5 or control antibody 11B7 and stimulated withFGF1 or FGF19. Erk phosphorylation was determined in a cell ELISA asdescribed in example 2.

FIG. 5. Antibody 10F10 inhibits Erk phosphorylation stimulated by FGF19in ZR-75-1 breast cancer cells. Experimental condition were similar tothose in FIG. 2.

EXAMPLES Example 1 Generation of the Antibodies

For generation of antibodies that bind specifically to FGFR4 arecombinant Glutathione-S-Transferase (GST) (Smith & Johnson, 1988)fusion protein comprising the human FGFR-4 extracellular domain (FGFR-4ex) was prepared. We used the cloning vector pSj26(mod) (Seiffert etal., 1999) that was designed for the eukaryotic expression and secretionof recombinant fusion proteins and was derived from the pcDNA3 cloningvector (Invitrogen, Groningen, The Netherlands) by inserting thecomplete DNA sequence coding for Schistosoma japonicumglutathione-S-transferase (GST) (Pharmacia Biotech, Freiburg, Germany)in the XhoI and ApaI sites of pcDNA3.

The extracellular domain of human FGFR-4 was PCR amplified using thefollowing primers:

sense: (SEQ ID NO: 21) 5′-GAATTCGCCACCATGCGGCTGCTGCTGGCCCTGTTG-3′,antisense: (SEQ ID NO: 22) 5′-CGAGGCCAGGTATACGGACATCATCCTCGAGTT-3′.

The PCR product was digested with EcoRI and XhoI and cloned intopSj26(mod). The resulting pSj26(mod)-FGFR-4ex expression plasmid wastransfected into 293 cells (ATCC CRL-1573) by the calcium phosphate DNAcoprecipitation method. Cells were grown in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% FCS. After selection with 1 mg/mlG418 (Sigma, Deisenhofen, Germany) for two weeks, surviving clones weretested for expression and secretion of the fusion protein by Westernblot analysis with antibodies against GST. High-expressing cells wereused to produce FGFR-4ex. Medium was collected from confluent culturesevery two days. One liter of collected medium was sterile filtered andincubated with 1 ml glutathione Sepharose (Pharmacia Biotech, Freiburg,Germany) overnight at 4° C. The Sepharose was separated and washed withphosphate-buffered saline (PBS). Elution was performed with 5 ml 10mmol/l glutathione at 20° C. Eluted fusion protein was dialyzed 1:10⁶(vol/vol) in PBS/10% glycerol. Protein concentration was determinedusing MicroBCA protein determination kit (Pierce, Rockford, Ill.).

Monoclonal antibodies were raised by injection of approximately 50 μg ofFGFR4-ECD-GST fusion protein both i.p. and subcutaneously into Lou/C orLong Evans rats using CPG2006 (TIB MOLBIOL) or Freund's incompleteadjuvant as adjuvants. After an 8-week interval, a final boost was giveni.p and subcutaneously 3 d before fusion. Fusion of the myeloma cellline P3X63-Ag8.653 with the rat immune spleen cells was performedaccording to standard procedures. Hybridoma supernatants were analyzedfor isotype and subclass by ELISA and tested by FACS for binding toCHO-FGFR4 cells.

A determination of nucleotide sequences encoding antibodies as generatedabove, was carried out. SEQ ID NO:1 and 2 show a nucleotide sequence ofthe heavy or light chain regions of the antibody 10F10. SEQ ID NO: 3 and4 show the nucleotide sequence of the heavy or light chain variableregions of the antibody 9A5. The amino acid sequences of the respectiveheavy and light chain regions are shown in SEQ ID NO: 5-8.

SEQ ID NO: 9-14 show the amino acid sequences HCDR1, HCDR2, HCDR3,LCDR1, LCDR2 and LCDR3 of antibody 10F10. SEQ ID NO:15-20 show the aminoacid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of antibody9A5.

Example 2 Anti-FGFR4 Antibodies 10F10 and 9A5 do not Bind to the OtherMembers of the Family, FGFR1-3

Recombinant FGFR1-4 ECDs or BSA were coated to Maxisorp plates (Nunc, x)at 12.5 nM (100 μl per well) at 4° C. over night. After one wash withwashing buffer (PBS pH 7.4, 0.05% Tween-20), plates were blocked with 1%BSA in washing buffer (blocking buffer) for 2 hours at room temperature.FGFR4 antibodies 10F10 and 9A5 as described in Example 1, non-bindingcontrol antibody 13F2 or anti-myc antibody 9E10 (Abcam) were added at 1μg/ml in blocking buffer (100 μl per well) and incubated for 2 hours atroom temperature. Plates were washed 6 times with washing buffer andincubated with the appropriate, POD-coupled secondary antibodies for 45minutes at room temperature. After 6 washes with washing buffer and onewash with PBS, POD activity was determined by incubation with 100 μl TMBPOD substrate (Calbiochem) per well for 5 minutes at room temperaturefollowed by addition of stop solution (250 mM HCl, 100 ml). Absorbancewas quantified in an ELISA reader at 450 nm (FIG. 1).

Example 3 Generation of Cells Overexpressing FGFR4

Appropriate FGFR-4 cDNAs were amplified from K562 cells (ATCC CCL-243),and subcloned into the Bluescript I KS vector (Stratagene) according tostandard protocols (Current Protocols). The FGFR4 cDNA was then clonedinto the pLXSN vector (Stratagene). The packaging cell line GF+E 86(Markowitz et al., 1988) that produces ecotrophic viruses wastransfected with this vector using calcium phosphate DNAcoprecipitation. The supernatant of transfected GF+E 86 cells wascollected and filtered through a 0.45 μm filter. Cells infected with thevector pLXSN alone were used as controls. For infection of rat L6myoblasts, which does not express detectable amounts of FGFR-4, cellswere incubated with viral supernatant for 24 h. After 48 h, medium wasreplaced with medium containing 400 μg/ml G418 and further selectedunder G418 for 14 days.

Accordingly, CHO cells expressing FGFR4 were generated by transfectionof parental CHO cells with FGFR4 cDNA cloned into the pcDNA3 vector.Clonal cells lines were generated by limited dilution. FGFR-4 expressionwas determined by western blot analysis.

Example 4 Determination of 10F10 and 9A5 Antibody Affinities in FACSScatchard Experiments on L6-FGFR4 Cells

L6-FGFR4 cells were harvested by incubation with 10 mM EDTA in PBS andresuspended at 6 million cells per ml in FACS buffer (PBS pH 7.4, 3%FCS, 0.1% NaN₃). In a round-bottom microtiter plate, 100 μl of cellsuspension were added to 100 μl of antibody solution containingantibodies 10F10 or 9A5 at concentrations between 31.25 and 0.01 μg/mlin FACS buffer. Antibody binding was allowed to proceed for 2 hours ice.Then, cells were washed twice with 250 μl FACS buffer per well, andresuspended in 200 μl of secondary antibody (anti-rat-PE; Jackson),diluted 1:50 in FACS buffer. After 45 minutes of incubation, cells wereagain washed twice in FACS buffer, then resuspended in 500 ml of PBS forFACS analysis. Analysis was carried out on a Beckman-Coulter FACS. Todetermine the apparent affinity constant K_(Dapp), mean fluorescencevalues were plotted against the ratio of mean fluorescence and thecorresponding antibody contration ([M]). The calculated K_(Dapp)resulted from the inverse slope of the straight line (FIG. 2).

Example 5 Anti-FGFR4 Antibodies 10F10 and 9A5 Inhibit FGF1-Induced FGFR4Tyrosine Phosphorylation

1.5×10⁶ L6-FGFR4 cells were seeded in a 10-cm culture dishes and thefollowing day starved for 24 hours in serum-free medium. Then, cellswere incubated with antibodies 10F10 or 9A5 at 10 μg/ml for 1 hour andstimulated with 10 ng/ml FGF1 (R&D Systems, x) for 10 minutes. Cellswere scraped off in 1 ml lysis buffer (50 mM Tris-HCl, pH 7.4, 1%Triton-X-100, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, aprotinin/leupeptin,pepstatin 1 μg/ml each, 1 mM Na₃VO₄, 1 mM NaF) on ice and centrifugedfor 10 minutes at 10,000×g, 4° C. 1 ml of the supernatant were incubatedwith 20 μl of a suspension of protein A-sepharose beads and 1 μg of thepolyclonal anti-FGFR4 antibody C16 (Santa Cruz) on a rotator wheel at 4°C. over night. The beads were washed three times with HNTG buffer (20 mMHEPES pH 7.5, 150 mM NaCl, 0.1% Triton-X-100, 10% glycerol) and boiledin 40 μl Laemmli buffer. After SDS PAGE, proteins were blotted onto anitrocellulose membrane, which was subsequently blocked and incubatedover night with anti-phospho-tyrosine antibody 4G10 (Upstate). After 3washes, the membrane was incubated in HRP-coupled secondary antibody for2 hours at room temperature and washed gain 3 times. Detection was doneusing the ECL system (GE Healthcare) (FIG. 3).

Example 6 Anti-FGFR4 Antibodies 10F10 and 9A5 Inhibit ErkPhosphorylation Stimulated by FGF1 or FGF19 in FGFR4-Transfected L6Myoblasts

L6-FGFR4 cells were seeded into a 96-well plate at a density of 10,000cells per well in DMEM/10% FCS. Cells were starved for 24 hours inserum-free medium, then antibodies 10F10, 9A5 or I11B7 were added at 10μg/ml in fresh, serum-free medium. After 1 hour of incubation, cellswere stimulated by addition of 10 ng/ml FGF1 or 600 ng/ml FGF19; FGF19had been purified by StrepTactin-affinity chromatography (IBA) afterexpression from HEK-293 cells as Strep-tagged recombinant protein. Forconstruction of the expression construct, the coding sequence of FGF19had been inserted into the vector pcDNA3, and HEK-293 had been stablytransfected using Lipofectamine 2000 (Invitrogen) and selection with 500μg/ml G418.

After stimulation, cells were fixed by addition of 4% formaldehyde inPBS and incubation for 1 hour at room temperature. After 2 times washingwith washing buffer (PBS pH 7.4, 0.1% Tween 20) for 5 minutes each, theplate was incubated with 100 μl of quenching buffer (1% H₂O₂ and 0.1%NaN₃ in washing buffer) per well. After two more washes, 100 μl ofblocking buffer (PBS, 0.5% BSA) were added per well and the plateincubated at 4° C. over night. The next day, anti-phosphoErk antibody(Cell Signaling Technologies) was added at a dilution of 1:3000 indilution buffer (PBS, 0.5% BSA, 0.05% Tween-20, 5 mM EDTA) and incubatedfor 4 hours at room temperature. Cells were washed 3 times with 200 μlwashing buffer per well and incubated with POD-conjugated secondaryantibody for 90 minutes at room temperature. After 3 washes with washingbuffer and one with PBS, 100 μl of TMB were added and the plateincubated for 20 minutes at room temperature. The absorbance at 450 nmwas measured after addition of 100 μl of stop solution per well in anELISA reader (FIG. 4).

Example 7 Antibody 10F10 Inhibits Erk Phosphorylation Stimulated byFGF19 in ZR-75-1 Breast Cancer Cells

ZR-75-1 cells were obtained from ATCC and routinely culture in RPMI/10%FCS. The experimental conditions for antibody incubation, stimulationand PhosphoErk-ELISA were identical to those described in Example 6. Itwas found that antibody 10F10 is capable of blocking FGF19 stimulatedErk phosphorylation. (FIG. 5).

LITERATURE

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1. An isolated antibody that binds to the extracellular domain of FGFR4or an antigen binding fragment thereof, wherein the antibody or antigenbinding fragment comprises at least one heavy chain amino acid sequencecomprising the following CDRs: a CDRH1 as shown in SEQ ID NO: 9, (b) aCDRH2 as shown in SEQ ID NO: 10, and (c) a CDRH3 as shown in SEQ ID NO:11, and at least one light chain amino acid sequence comprising thefollowing CDRs: a CDRL1 as shown in SEQ ID NO: 12, (e) a CDRL2 as shownin SEQ ID NO: 13, and a CDRL3 as shown in SEQ ID NO:
 14. 2. The antibodyof claim 1, which comprises a heavy chain amino acid sequence of SEQ IDNO: 5 or at least the variable domain thereof or an amino acid sequencehaving an identity of at least 90% thereto, and a light chain amino acidsequence of SEQ ID NO:6 or at least the variable domain thereof or anamino acid sequence having an identity of at least 90% thereto.
 3. Theantibody according to claim 1, which is a monoclonal antibody, arecombinant antibody, a humanized antibody, a chimeric antibody, amultispecific antibody, or an antigen binding fragment thereof.
 4. Theantibody according to claim 1, which is a Fab fragment, a Fab′ fragment,a F(ab′)₂ fragment, a Fv fragment, a diabody, or a single chain antibodymolecule.
 5. The antibody according to claim 1, which is of the IgG1-,IgG2-, IgG3- or IgG4-type.
 6. The antibody according to claim 1, whichis coupled to a labeling group.
 7. The antibody according to claim 6,wherein the labelling group is a radioisotope or radionuclide, afluorescent group, an enzymatic group, a chemiluminescent group, abiotinyl group, or a predetermined polypeptide epitope.
 8. The antibodyaccording to claim 1, which is coupled to an effector group.
 9. Theantibody according to claim 8, wherein the effector group is aradioisotope or radionuclide, a toxin, or a therapeutic orchemotherapeutic group.
 10. A pharmaceutical composition comprising anantibody according to claim
 1. 11. The pharmaceutical composition ofclaim 10 comprising pharmaceutically acceptable carriers, diluentsand/or adjuvants.
 12. The pharmaceutical composition according to claim10, optionally comprising a further active agent.
 13. A kit comprisingan antibody according to claim
 1. 14. The kit according to claim 13,further comprising a further antineoplastic agent.
 15. An isolatedantibody that binds to the extracellular domain of FGFR4 or an antigenbinding fragment thereof, wherein the antibody or antigen bindingfragment comprises at least one heavy chain amino acid sequencecomprising the following CDRs: (a) a CDRH1 as shown in SEQ ID NO: 9, (b)a CDRH2 as shown in SEQ ID NO: 10, and (c) a CDRH3 as shown in SEQ IDNO: 11, and at least one light chain amino acid sequence comprising thefollowing CDRs: (d) a CDRL1 as shown in SEQ ID NO: 12, (e) a CDRL2 asshown in SEQ ID NO: 13, and (f) a CDRL3 as shown in SEQ ID NO: 14,wherein said antibody reduces or blocks FGFR4 phosphorylation byreducing or blocking FGFR4-mediated signal transduction; said antibodybinds to the extracellular domain of FGFR4 at a location which reducesor blocks ligand binding; said antibody inhibits enough FGFR4 activityto reduce or block cell proliferation; and said antibody inhibits enoughFGFR4 activity to reduce or block cell migration.